Endocytosis brings essential nutrients and other molecules into cells, regulates signaling by cell-surface receptors, and modulates interactions with the extracellular environment. Our general goal is to understand how endocytosis affects phenotypes and disease. Disabled 2 (Dab2) is a "phosphotyrosine-binding" (PTB) domain protein that binds to clathrin and other endocytic proteins and localizes to clathrin-coated pits. We have now shown that Dab2 knockout inhibits endocytosis in vivo and in vitro. Dab2 functions as a clathrin- associated sorting protein (CLASP) for low density lipoprotein receptors (LDLRs). Unlike all previously characterized CLASPs, Dab2 function is independent of AP-2. We also found that Dab2 depletion from normal cells affects adhesion, migration and integrin endocytosis. Dab2 is known to be strongly down-regulated in many different human carcinomas, particularly ovarian and mammary tumors. Loss of Dab2 allows cancer cells to resist anoikis (detachment-induced apoptosis). We would like to understand how Dab2 could cause such phenotypic changes through its role in endocytosis. The proposed research builds on a new technology that allows us to identify receptors trafficked by a given adaptor protein. This approach has revealed that integrin endocytosis requires Dab2. Changes in integrin levels correlate with Dab2-induced changes in focal adhesion turnover during cell spreading and migration. Dab2 is the first adaptor protein found for integrin endocytosis. We are now in a unique position to study the importance of integrin endocytosis for cell adhesion, cytoskeletal organization and motility. We hypothesize that Dab2-dependent cell surface proteins may be up-regulated on the surface when Dab2 is absent, and may be responsible for reduced migration and increased anoikis-resistance of Dab2-deficient cells. We now propose two aims, one global and one specific. In the first Aim, we will identify cargoes requiring different PTB adaptor proteins, Dab2, ARH and Numb, determine how cargoes are selected, and identify internalization signals. We will explore how specific cargoes relate to the cellular phenotypes of Dab2, ARH and Numb, including the effects of Dab2 re-expression in cancer cells. In the second Aim, we will investigate Dab2-mediated endocytosis of integrins. We will ask how integrins are recognized for endocytosis;whether Dab2 mediates basal or adhesion-dependent internalization;whether Dab2-mediated integrin uptake is regulated by growth factors or the cytoskeleton;how integrin endocytosis relates to cell migration, adhesion and cytoskeletal remodeling. We will test whether cancer cells in which Dab2 is down-regulated have increased surface levels of integrins and the role of integrin up-regulation in the cancer phenotype. These studies will enhance our understanding of how receptors and integrins are selected for endocytosis, and the role of cell surface proteins in cell biology and the progression of cancer. Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility. PUBLIC HEALTH REVELANCE: Proper interactions between cells and their environment are mediated by the outer surface of the cell, which is continually renewed by new proteins coming from, and old proteins returning to, the inside of the cell. Defects in internalization of surface proteins cause defects in development and are important in cancer. This application proposes to understand the mechanisms by which cell surface proteins are recognized for internalization. The results will be informative regarding changes in cancer cells that affect cell survival and motility.